Structural Organization Of G-protein Coupling Systems

G 蛋白偶联系统的结构组织

• 批准号: 6842472
• 负责人: ROBERT VICTOR REBOIS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6842472
• 关键词: G protein; adenylate cyclase; beta adrenergic receptor; binding proteins; biological signal transduction; bioluminescence; cyclic AMP; enzyme activity; fluorescence resonance energy transfer; genetic transcription; green fluorescent proteins; guanine nucleoside; human tissue; luciferin monooxygenase; membrane reconstitution /synthesis; protein protein interaction; protein structure; receptor coupling; tissue /cell culture

G protein-mediated signal transduction systems are involved in the responses of organisms and their constituent cells to a wide variety of stimuli including light, gustants, odorants, hormones, and neurotransmitters. The nature of the response can be equally diverse varying from changes in gene transcription to altered transmembrane ion permeability. The three core components of this system are the heptahelical receptors, heterotrimeric G proteins and effector molecules which must interact in order to convey information from one component to the next. The prevailing view has been that these interactions are the result of random collisions between signaling molecules that move about freely in the plasma membrane. However, accumulating data has provided evidence that signaling molecules are organized into macromolecular complexes on the cell surface. In order to elucidate the spacial arrangement of the proteins that make up these signaling complexes a technique known as bioluminescence resonance energy transfer (BRET) is being used to investigate protein-protein interactions between the various signaling molecules in living cells. The signaling molecules are expressed in transfected mammalian cells as fusion proteins tagged with either the bioluminescent protein luciferase (RLuc) or green fluorescent protein (GFP). If the tags are brought into juxtaposition by a stable protein-protein interaction between two signaling molecules, BRET occurs because light emitted by the RLuc tag will be absorbed by the GFP tag which then fluoresces. For these studies, we are using the prototypical beta2-adrenergic receptor (b2AR) signaling system that consists of the b2AR, the stimulatory heterotrimeric G protein (Gs), and the effector adenylyl cyclase (AC). Agonist stimulation of the b2AR results in the Gs-mediated stimulation of AC leading to the production of cyclic AMP. When the b2AR, G protein subunits and AC are tagged with GFP or RLuc these signaling molecules retain their biological activity. In HEK 293 cells co-expressing these tagged proteins BRET occurred between GFP-tagged G protein subunits and both the b2AR-RLuc and AC-RLuc. We have also shown that BRET occurs between the b2AR-GFP and AC-RLuc. These date indicate that the b2AR, Gs and AC form a complex in living cells. This complex is present in the absence of hormone stimulation, and BRET persists in the presence of hormone suggesting that these complexes exist in both the basal state and during signal transduction, thus providing support for the evolving view that G protein-mediated signaling systems exist as organized complexes that contribute significantly to the specificity and efficacy of the signal transduction process. In addition to forming complexes with down stream signaling molecules, heptahelical receptors interact with each other to form receptor dimers. As the receptor's polypeptide chain passes back and forth through the plasma membrane it creates loops that extend into the extra- and intracellular spaces and deposits the C-terminus within the cytoplasmic milieu. The second and third intracellular loops are both critical for interaction with downstream signaling components, and the lack of either domain renders the receptor unable to mediate signal transduction despite retention of the ability to bind ligand. It has been hypothesized that receptor dimerization results in intermolecular interactions in which a portion of one receptor is associated with the complementary part of the other, thus forming a domain that interacts with the heterotrimeric G proteins. To test this hypothesis two signaling deficient b2ARs (one that is missing the second intracellular loop and one that is missing the third intracellular loop) are being co-expressed in HEK 293 cells in order to determine if a fully functional b2AR can be reconstitute from the two mutated receptors.

G蛋白介导的信号转导系统参与了生物体及其成分细胞对各种刺激的反应，包括光，胶，气味，激素和神经递质。响应的性质可能是从基因转录的变化到变化的跨膜离子渗透性的不同。该系统的三个核心组成部分是七螺旋体受体，异三聚体G蛋白和效应分子，它们必须相互作用才能从一个成分传达到下一个成分。流行的观点是这些相互作用是在质膜中自由移动的信号分子之间随机碰撞的结果。但是，累积数据提供了证据，表明信号分子被组织到细胞表面上的大分子复合物中。为了阐明构成这些信号传导复合物的蛋白质的空间排列，一种称为生物发光谐振能量转移（BRET）的技术正在用于研究活细胞中各种信号分子之间的蛋白质 - 蛋白质相互作用。信号分子在转染的哺乳动物细胞中表示为用生物发光蛋白荧光素酶（RLUC）或绿色荧光蛋白（GFP）标记的融合蛋白。如果标签通过两个信号分子之间稳定的蛋白质 - 蛋白质相互作用并并置，则会发生BRET，因为RLUC标签发出的光将被GFP标签吸收，然后将其吸收。在这些研究中，我们使用了由B2AR，刺激性异三聚体G蛋白（GS）和效应子腺苷酸环化酶（AC）组成的典型β2-肾上腺素能受体（B2AR）信号系统。 B2AR的激动剂刺激导致GS介导的AC刺激，从而导致循环AMP产生。当B2AR，G蛋白亚基和AC用GFP标记或RLUC时，这些信号分子保留其生物学活性。在HEK 293细胞中，共表达这些标记的蛋白质的Bret发生在GFP标记的G蛋白亚基与B2AR-RLUC和AC-RLUC之间。我们还表明，BRET发生在B2AR-GFP和AC-RLUC之间。这些日期表明B2AR，GS和AC在活细胞中形成复合物。这种复合物存在在没有激素刺激的情况下存在，而在存在激素的情况下，BRET持续存在表明这些复合物在基础状态和信号转导期间都存在，从而为不断发展的观点提供了支持G蛋白介导的信号系统作为有组织的络合物而存在的支持，从而对信号传输过程的特异性和效率显着影响。 除了形成下流信号分子的络合物外，七旋hel型受体相互相互作用以形成受体二聚体。当受体的多肽链通过质膜来回传递时，它会产生循环，从而延伸到细胞内和细胞内空间，并沉积在细胞质环境中的C末端。第二和第三个细胞内回路对于与下游信号传导分量的相互作用至关重要，并且缺乏任何一个结构域使受体无法介导信号转导，尽管保持了结合配体的能力。已经假设受体二聚化导致分子间相互作用，其中一个受体的一部分与另一个受体的互补部分相关，从而形成了与异核三聚体G蛋白相互作用的结构域。为了检验这一假设，在HEK 293细胞中共表达了两个缺乏B2AR（缺少第二个细胞内环和一个缺少第三个细胞内环的一个缺乏的B2AR），以确定是否可以从两个突变受体中重新构成功能齐全的B2AR。